1. Field of the Invention
The present invention relates to a method for obtaining a plant with a lasting resistance to a pathogen. The invention also relates to a plant in which two or more resistance genes to the pathogen are present, in addition to seeds and progeny of this plant, and progeny thereof.
The invention relates particularly to a method for obtaining a cultivated lettuce plant (L. sativa) with a lasting resistance to Bremia lactucae. The invention also relates to DNA-markers which are specifically linked to a resistance gene to Bremia lactucae. The invention further relates to a cultivated lettuce plant (L. sativa) in which two or more Dm-resistance genes are present, and to seeds and progeny of this plant, and progeny thereof.
2. Description of the Related Art
The disease which is caused by the fungus Bremia lactucae Regel is known as downy mildew. Downy mildew occurs worldwide and represents a great problem for both the yield and quality of cultivated lettuce. The fungus can infect the lettuce plant at any stage of growth, after which the first symptoms of downy mildew consist of the appearance of chlorotic yellow spots on the leaf surface. Within 24 to 48 hours a white fluffy fungus growth then becomes visible an the lower leaf surface as an indication of spore formation. During the infection the lesions become increasingly larger and more chlorotic until the leaves become completely brown.
Bremia lactucae is one of the so-called Oomycetes, a class of relatively primitive fungi. Other known fungi of this group are for instance Phytium and Phytophtora. The fungus B. lactucae contains different physiological species (“physios”) and is host-specific. Bremia lactucae is known as a very variable pathogen. New physios occur relatively frequently through mutation of the avirulence genes during the spore formation preceding the propagation of B. lactucae. 
Within the Lactucae genus, to which the cultivated lettuce (Lactuca sativa) belongs, there are different species which are resistant to Bremia lactucae Regel. The resistance is based in most cases on qualitative genes, known as Dm-resistance genes (Dm=Downy mildew). The resistance mechanism is known as a gene-for-gene working principle based on the specific interaction between products of the Dm-resistance gene and the pathogen-specific avirulence gene, which results in resistance of the lettuce plant (Michelmore et al., Plant Pathology 33, 301-315, 1984). This resistance mechanism has also been demonstrated for diverse other resistance genes in different other plant species (Michelmore et al., Genome Research, 8, 1113-1130, 1998).
A large number of Dm-resistance genes have already been identified which can bring about resistance to specific physics of Bremia lactucae Regel. Genetic research has shown that these Dm-resistance genes often occur clustered in groups on the same chromosome. Four such linking groups on different chromosomes in the genome of lettuce have been demonstrated which contain different Dm-resistance genes (Farrara et al., Plant Pathology 36, 499-514, 1987). Newly identified Dm-genes can often be classified into one of the known resistance linking groups.
A major problem however is that Bremia lactucae physics continue to occur which “break down” the resistance resulting from the known Dm-resistance genes in the present cultivated lettuce varieties. This implies that Bremia lactucae physios occur to which there is no resistance in present cultivated lettuce varieties. Resistance genes can however sometimes still found in old lettuce cultivars, but particularly in wild Lactucae species related to cultivated lettuce, such as for instance L. virosa and L. serriola. A number of broad-spectrum Dm-resistance genes have been identified with a resistance to all tested Bremia physios.
Dm-resistance genes from old lettuce cultivars or from wild lettuce species can be crossed into cultivated lettuce to once again obtain resistance. Crossed-in Dm-resistance genes have been demonstrated in conventional manner by means of an artificial Bremia lactucae disease test. For this purpose a number of leaf punches—(diameter 18-20 mm) or seedlings of the lettuce plant are inoculated with different physios of B. lactucae. After 10 to 14 days the degree of development and sporulation on the punches/seedlings is then examined. On the basis hereof it is possible to judge whether a tested lettuce plant or improved line is resistant or susceptible to the tested B. lactucae physios.
When it is known that two or more new Dm-resistance genes occur in different linking groups, these resistance genes can be brought together (‘stacked’) in a cultivated lettuce plant by crossing-in, thereby reducing the danger of the resistance being broken down. Stacking of a plurality of qualitative broad-spectrum Dm-resistance genes from different linking groups can however not be carried out with the conventional Bremia lactucae disease test because, when one qualitative Dm-resistance gene is present, total resistance is already detected in the disease test and the possible presence of a second broad-spectrum Dm-resistance gene will therefore not be detected. It is therefore not possible to select precisely those plants in which two or more qualitative broad-spectrum Dm-resistance genes are present and thus obtain plants with a lasting resistance to B. lactucae. 
It is therefore desirable for a method to be developed with which, after crossing of qualitative resistance genes into a plant, those plants can be identified and selected in which two or more resistance genes are present.